Characterization of 3'-phosphoadenosine 5'-phospho[35S]sulfate transport carrier from rat brain microsomes.
3'-Phosphoadenosine 5'-phospho[35S]sulfate [( 35S]PAPS) specific binding properties of rat brain tissue were studied. [35S]PAPS specific binding was optimal at pH 5.8 in either Tris-maleate or potassium phosphate buffers. Association was maximal at low temperature, reaching equilibrium in 20 min. Dissociation was rapid, with a dissociation time of 80 s. Scatchard analysis of [35S]PAPS specific binding was consistent with a single site having a KD of 0.46 +/- 0.06 microM and a Bmax of 20.8 +/- 2.0 pmol/mg of protein. Low concentrations of Triton X-100 (0.025%) were effective in increasing the number of binding sites to a Bmax of 44.5 +/- 4.6 pmol/mg of protein without affecting the affinity. [35S]PAPS specific binding was enriched in crude synaptic membranes (P2) and microsomes (P3). Regional distribution of [35S]PAPS specific binding was quite homogeneous in all brain structures studied. The pharmacological profile of [35S]PAPS specific binding in rat brain microsomes was consistent with a membrane protein having a high selectivity for the 3'-O-phosphoryl group substitution on the ribose moiety. Thus, 3'-phosphoadenosine 5'-phosphate was more potent than 2'-phosphoadenosine 5'-phosphate in competing for [35S]PAPS specific binding. Adenosine 5'-phosphosulfate was a good inhibitor of [35S]PAPS specific binding. ATP and ADP were also good displacers. Dipyridamole, a highly selective marker for adenosine uptake sites, was ineffective. 4,4-Diisothiocyanostilbene-2,2-disulfonic acid, the chloride transporter inhibitor, showed an IC50 of 36 +/- 5.1 microM for inhibition of [35S]PAPS specific binding. 2,6-Dichloro-4-nitrophenol had a low selectivity in competing for the [35S]PAPS binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
Vargas
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Laboratoire de Physiologie, Université René Descartes, Paris, France.
D000227:Adenine Nucleotides / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D003080:Cold Temperature; D006863:Hydrogen-Ion Concentration; D008297:Male; D008861:Microsomes / Q000378:metabolism*; D010724:Phosphoadenosine Phosphosulfate / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013462:Sulfur Radioisotopes; D013574:Synaptosomes / Q000378:metabolism; D014018:Tissue Distribution
D000227:Adenine Nucleotides; D002352:Carrier Proteins; D013462:Sulfur Radioisotopes; D010724:Phosphoadenosine Phosphosulfate